PerspectivePARASITOLOGY

A Requiem for Chloroquine

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Science  04 Oct 2002:
Vol. 298, Issue 5591, pp. 74-75
DOI: 10.1126/science.1077573

Chloroquine (CQ) has historically been the mainstay of malaria treatment, particularly in the worst affected regions of sub-Saharan Africa. The recent development of widespread CQ resistance in Plasmodium falciparum, the most dangerous of the four malaria parasite species, has contributed significantly to escalating mortality rates in Africa (1) and to the resurgence of malaria as an immediate public health priority (2). Several pressing scientific questions have emerged within the context of this humanitarian disaster: What is the molecular basis for CQ resistance, and how has this influenced the dynamics of resistance? Why did CQ remain effective for 20 years, yet its immediate replacement sulfadoxine-pyrimethamine (SP) last less than 5 years? Has the widespread deployment of CQ jeopardized the use of other drugs targeting the same parasite biochemical pathways? As reported on page 210 of this issue, Sidhu et al. (3) have obtained data relevant to all three questions by creatively exploiting the pfcrt gene, which encodes a putative transporter protein in the digestive vacuole membrane of the malaria parasite. They replaced the endogenous pfcrt gene in a CQ-sensitive strain of P. falciparum with a pfcrt gene from each of three CQ-resistant strains. All such replacement strains (“constructs”) showed CQ resistance in vitro, demonstrating that pfcrt mutations are sufficient, within their selected genetic background, to encode resistance. Reduced levels of pfcrt gene expression in the constructs also showed that up-regulation of pfcrt is not required for resistance. Next, the authors investigated cross-resistance between CQ and other antimalarial drugs.

Evolutionary dynamics of CQ resistance.

The solid line shows the serum concentrations of CQ after treatment of a typical human host. Three consecutive daily doses are given, after which the CQ concentration declines to subtherapeutic levels around day 10 (11). Resistance to CQ probably arises through the sequential accumulation of mutations (numbers 1 to 7) encoding gradually increasing drug tolerance. Critically, the malaria parasites remain susceptible to therapy because drug levels after treatment exceed their tolerance. Eventually, a mutation occurs (number 8) that enables the parasites to survive post-therapy drug levels.

CREDIT: KATHARINE SUTLIFF/SCIENCE

Previous work from this and other groups has implicated eight or nine different pfcrt mutations in the development of CQ resistance (4). The sequential accumulation of these mutations plausibly explains the observed genetics and epidemiology of CQ resistance (see the figure). So why did CQ last so much longer than SP as a frontline antimalarial? First, four sequential mutations in the dhfr gene—which encodes dihydrofolate reductase, an enzyme essential for parasite folate metabolism and targeted by the drug pyrimethamine—appear sufficient for SP resistance (5). These four mutations accumulate much faster than the nine required for CQ resistance. Second, CQ persists at therapeutically useful concentrations for a much shorter period than SP, leading to lower selection pressures for resistance (6). Third, CQ resistance may involve genes other than pfcrt, such that sexual recombination during the malaria life cycle breaks down genetic combinations, slowing resistance (7, 8). The putative involvement of other genes remains controversial. Sidhu et al. show that pfcrt alone is sufficient to encode resistance, but this only holds for the genetic background of their selected host strain (50% of which was derived from a CQ-resistant strain). Field studies are similarly inconclusive: pfcrt Lys76 → Thr (K76T) mutations appear to be a prerequisite for parasites to survive drug treatment, but many pfcrt K76T parasite infections disappear after drug treatment. The interpretation of this simple observation remains unclear: It may implicate other genes, or it may simply represent human immunity eradicating truly drug-resistant infections. The Sidhu et al. paper contains the technology required to perform the definitive test of introducing an identical pfcrt CQ-resistant construct into a series of different genetic backgrounds; such results are eagerly anticipated.

The realization that the long therapeutic life-span of CQ was likely to be the exception rather than the rule led the World Health Organization to recommend that all new antimalarial treatments be deployed as drug combinations. The efficacy of drug combination strategies has been demonstrated mathematically and by the success of combination therapy for HIV and tuberculosis. A fundamental requirement of combination therapy is that the genetic basis of resistance differs for each drug because even a small amount of cross-resistance dramatically increases the rate at which resistance evolves (9), severely limiting the likely therapeutic life-span of the drug combinations. In practice, the dearth of new antimalarial drugs means that existing drugs will be redeployed in combination with the relatively new antimalarial artesunate, a derivative of artemisinin. This has raised fears that cross-reactivity may have evolved because quinolines (such as CQ) and artemisinins both kill the malaria parasite by interfering with its heme detoxification pathway. Reassuringly, Sidhu et al. demonstrate that as resistance to CQ evolves, the parasites become more susceptible to artemisinins.

Resistance to CQ probably arises through the sequential accumulation of mutations (see the figure). The first mutations spread because they confer increased tolerance to CQ on parasites, enabling them to infect humans sooner after drug treatment—for example, mutation 4 allows parasites to infect people 6 days after treatment rather than 7 days. The relatively rapid elimination of CQ means that these are rather weak selective forces (6) and that the spread of these first mutations will be slow. Eventually, mutation 8 arises, which allows the parasite to survive therapeutic levels of CQ. Once above this threshold, the selective advantage conferred by this mutation becomes enormous and the pfcrt haplotype (now containing several sequentially acquired mutations) spreads rapidly across geographic regions where CQ is in common use. This appears to have occurred four times for CQ resistance: twice in South America, once in southeast Asia, and once in Papua New Guinea (see the viewpoint by Wellems on page 124) (10). The mutations may not have equal effects: mutations K76T and Ala220 → Ser (A220S) appear to be the most reliable markers predicting CQ resistance. There are three plausible explanations for this: (i) If the mutations can be acquired in any sequence and K76T and A220S have large effects, then they will have a stronger correlation with resistance; the problem with this argument is that they rarely, if ever, occur alone and invariably occur with other “lesser” pfcrt mutations. (ii) Mutation acquisition may follow a set sequence with K76T and A220S near its end. (iii) These are the pharmacologically important mutations. The other mutations are optional—they may have a small effect on CQ tolerance, or compensate for impaired protein activity after the acquisition of the K76T or A220S mutations, or encode resistance during the transmission stages of the malaria life cycle.

In retrospect, CQ was a wonder drug. Cheap, safe, and effective against one of the major human killer diseases, it remained effective for 20 years in Africa amid the chaotic clinical setting of underdosing, noncompliance, and its indiscriminate use in treating all fevers. Hundreds of millions of treatment courses were deployed annually in Africa alone, and in many areas most of the population have detectable circulating CQ. It is hard to envisage any other drug lasting so long under these circumstances, especially since resistance finally arrived from southeast Asia rather than arising in Africa itself. Now it appears that the application of modern genetic technology may enable CQ to leave one more valuable legacy: a detailed genetic, clinical, and epidemiological epitaph that can be used to inform the deployment of its successors.

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